The present invention relates to nucleic acid hybridization assays which are useful as a means of locating specific nucleic acid sequences. Examples of nucleic acid sequences are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences. The molecular subunits of both DNA and RNA are called nucleotides which are linked together to form long polynucleotide chains. Each nucleotide subunit is made of a sugar moiety, a phosphate moiety and a base moiety. It is the sequential ordering of the base moieties [adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)]that contains DNA or RNA's genetic information. The ordering of these base moieties in a polynucleotide chain and the tendency of the bases to attract and bond with other specific base moieties, is exploited by this invention to locate, detect and isolate specific DNA or RNA sequences.
DNA normally contains two polynucleotide strands twisted about one another lengthwise in a helical manner resembling a ladder where the sides are made of identical sugar (deoxyribose) and phosphate molecules while the rungs are made up of bases extending out from each strand, held together by weak attractive forces. In DNA, the base thymine on one strand always pairs with the base adenine on the opposing strand, and the base guanine always pairs with the base cytosine. This is called complementary base pairing.
RNA is also a polynucleotide strand. However, the sugar moiety is ribose (versus deoxyribose in DNA) and the bases are adenine, guanine, cytosine and uracil. In RNA, the base uracil on one strand always pairs with the base adenine on the opposing strand, and the base quanine always pairs with the base cytosine. Although RNA can pair with either a complementary strand of RNA or DNA, it is normally single stranded so does not form a helical structure.
The present invention is founded, in part, upon the technique that single stranded nucleic acid sequences can be combined, or hybridized, under appropriate conditions with complementary single stranded nucleic acid sequences to form double stranded molecules. This technique was developed as a means for detecting and/or and isolating particular nucleic acid sequences of interest. It has increased in popularity during recent years in its application for detecting the presence of the DNA or RNA within such pathogens as viruses, bacteria, or other microorganisms and therefore the presence of these pathogens themselves. The technique can also be used for other purposes such as to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders (for example in sickle cell anaemia and thalassaemia), and to detect cancerous cells. Several applications have been developed for the microbiological analysis of clinical, food, environmental, and forensic samples. A general review of the technique and its present and future significance is provided in Biotechnology (August 1983), pp. 471-478 which is incorporated herein by reference.
The following definitions are provided to facilitate an understanding of the present invention. The term `probe` refers to a nucleic acid sequence of which there are at least three types: the primary probe, the amplification probe, and the labelling probe. The primary probe contains at least one nucleic acid sequence that is complementary (or will base pair) to some portion of a nucleic acid sequence on the target DNA or RNA molecule of interest. The amplification probe contains sequences that are complementary to some sequences on the primary probe, and contains a region that is typically of at least one type of repeating sequence unit. The labelling probe contains sequences complementary to one of the repeating sequence units, in addition to a chemical label. Labels are detectable chemical groups, either radioactive molecules or non-radioactive molecules and can include: radioactive isotopes; enzymatically active groups such as horse radish peroxidase; fluorescent agents; chemiluminescent agents; precipitating agents; and/or dyes. The term `signal` is used loosely to indicate the detectable characteristic of a detectable chemical group, which can include: a change in the light adsorption characteristics of a reaction solution resulting from enzymatic action of an enzyme attached to a labelling probe acting on a substrate; the color or change in color of a dye; fluorescence; phosphorescence; radioactivity; or any other indicia that will be evident to one skilled in the art.
The amplification probe is so named because it is used to cause many detectable chemical labels to become attached to one probe-target complex, such that the resulting signal is amplified in direct proportion to the number of labelled probes that hybridize to the amplification probe. If the amplification probe were to contain only one sequence unit that comprises sequences compatible to the labelling probe, only one labelling probe would become attached to the probe-target complex, and the signal would not be amplified. However, the amplification probe disclosed in the present invention contains typically five or more sequence units (also referred to as repeatmers) that are compatible to the labelling probe, such that five labelling probes will attach to one probe-target complex, resulting in five times the amount of detectable chemical label signalling the presence of one probe-target complex; thus, the indication that one probe-target complex was formed will be amplified five times. Moreover, if the amplification probe contains sixteen sequence units that are combatable to the labelling probe, sixteen labelling probes will attach to one probe-target complex, resulting in sixteen times the amount of detectable chemical label signalling the presence of one probe-target complex, the indication that one probe-target complex was formed will thereby be amplified sixteen times. The degree of amplification is optional and can be manipulated by the design and construction of the amplification probe as described herein.
One objective of a nucleic acid hybridization assay is to detect the presence of a specific nucleic acid sequence (the target sequence) in a given sample by contacting the sample with a complementary nucleic acid sequence (the probe) under hybridising conditions and observing the formation or absence of any probe-target complexes. The probe-target complex can be detected directly by a label attached to the probe. The complex can also be detected indirectly through such techniques as the hybridization of another nucleic acid sequence conjugated to a label or by the binding of an antibody labelled with a detectable chemical group.
One detection strategy currently employed in the art is exemplified by PCT Application 84/03520 and EPA 124221 which use an enzyme labelled nucleic acid sequence to detect the probe-target complex by hybridization to complementary sequences on the tail of the probe. For example, the Enzo Biochem "Bio-Bridge" system uses a biotin molecule conjugated to a poly(A) tail (a nucleic acid sequence comprised solely of adenine nucleotides) as the detection system following hybridization of a DNA probe possessing a poly(T) tail (a nucleic acid sequence comprised solely of thymine nucleotides) to the target DNA sequence.
In order to employ such a technique as an assay, one must be able to detect the presence or absence of probe-target complexes with a high degree of sensitivity. The sensitivity of a nucleic acid hybridization assay is determined primarily by the detection limit of the label to demonstrate the formation of the probe-target complex against back-ground noise and/or false-positives. Different strategies have been employed to improve the sensitivity of nucleic acid hybridization assays, which can be classified into four broad categories: 1) separation of the probe-target complex; 2) target amplification; 3) probe amplification; 4) multiple labelling, or combinations thereof.
Some nucleic acid hybridization assays involve immobilization of the target sequence on a solid support followed by washing away the remainder of the reaction mixture. This first category involves techniques that attempt to either immobilize the target sequence before adding a label probe or use an immobilized labelled probe to capture the target nucleotide sequence. Alternatively, techniques have been developed that immobilize the probe-target complex after its formation. For example, EPA Publication No. 0225807 discloses a nucleic acid hybridization assay in which the probe-target complex is removed from solution by hybridization with a complementary solid-supported capture probe. The solid phase complex is then detected by subsequent hybridization to a labelled probe. Generally, procedures attempting to immobilize the probe-target complex at this stage using a nucleic acid sequence suffer from the fact that proteins and other materials in the heterogeneous sample may have a higher tendancy to interfere with the immobilization of the nucleic acids. Furthermore, the sensitivity is low as the label to target ratio is 1:1.
A second category of strategies involves increasing the sensitivity of a nucleic acid hybridization assay through target amplification. An example of target amplification entails assaying for ribosomal RNA (rRNA) of a microorganism rather than chromosomal DNA. Since rRNA is present in any given cell at 10.sup.4 times higher concentration than DNA, the number of possible probe-target complexes increases, thereby increasing the probability of detecting the target organism. Alternatively, the polymerase chain reaction (PCR.TM.) described in U.S. Pat. Nos. 4,683,105 and 4,683,202 has been used to amplify target nucleic acid sequences. The advantages and limitations of this technique has been reviewed by Gyllensten (Biotechniques 7, 700-706, 1989, incorporated herein by reference). For example, this transcription-based amplification system can produce a 2-5 million-fold amplification of a RNA target after 4 cycles (Lizardi et al., Biotechnology 6, 1197-1202, 1988). However, this technique suffers from such problems as excessive noise, false positives, requires considerable technical expertise, and relatively expensive instruments and reagents (Walcott et al. Food Protein 54:387-401, 1991).
A third category of strategies for increasing the sensitivity of a nucleic acid hybridization assay entails probe amplification, by employing a combination of primary probes. Examples of this method are disclosed in U.S. Pat. Nos. 4,731,325 and 4,868,105 wherein techniques describe the use of more than one probe that binds to the target nucleic acid sequence. A further example is found in U.S. Pat. No. 4,868,105, where the labelled secondary probes hybridizes to the multiple primary probes bound to the target nucleic acid sequence.
Finally, some have attempted to employ multiple probes in a cascading or sandwich fashion as a strategy for amplifying the signal. These methods fall under the fourth category of signal amplification because they result in the attachment of multiple labelling groups to the primary probe-target complex. It is within this category that the present invention could be said to reside.
An early attempt to develop strategies within this fourth category is exemplified by PCT Application WO 90/13667 which describes an amplified solution-phase sandwich nucleic acid hybridization assay for the hepatitis B virus nucleic acid sequence in which the analyte is hybridized in solution with sets of amplifier probe polynucleotides and capture probe polynucleotides each have a first segment that is complementary to the target nucleic acid; furthermore, the amplifier probe has a second segment that is complementary to a unit of a polynucleotide multimer whereas the capture probe has a second segment that is complimentary to a polynucleotide bound to a solid phase. The resulting product is reacted with the polynucleotide bound to a solid phase and then with the multimer. The multimer probe is a chemically cross-linked single stranded oligodeoxyribonucleotide star-structured complex with arms possessing sequences complementary to the primary probe. Detection occurs when the bound materials are reacted with a labelled probe complementary to the polynucleotide units of the multimer.
In spite of the limited use of this strategy for detecting hepatitis B virus, the difficulties one would face devising the assay reagents are manifold, especially for general use. Constructing the chemical cross-linking in the secondary probe involves high levels of technical expertise as well as careful chemical modification of these polynucleotides in order to bring about the desired cross-linking. Overloading the star-like structures of the secondary probe can lead to steric hindrance between the star-like structures as well as between the anchoring arms attached to the solid phase. Furthermore, two sets of primary probes, both requiring the sequences complementary to the target nucleic acid, demand synthesis of probes designated to only one given target which is the case for a consensus hepatitis B virus double-stranded region sequence based on a multiplicity of hepatitis B viral subtypes. With these methods the cost of preparing probe reagents becomes significantly elevated eventually reflecting in the overall cost of the assay or the diagnostic kit. In short, this strategy is technically complex and the probe reagents are not applicable to a large variety of targets. Therefore, there remains a general need for a simpler system of signal amplification.
In yet a further method, Canadian Patent Application 2,039,517 provides a method for amplifying a signal wherein the amplification is obtained through use of a bridging nucleic acid sequence which can hybridize to the primary probe and to a developer nucleic acid sequence. This method entails hybridizing the primary probe to the target sequence, followed by exposure to the bridging sequence, followed by exposure to a first developer molecule, and finally followed by a second developer molecules is labelled, and the labelling can be detected in the developer chain. Again, the major limitation of this strategy is its complexity and there still remains a need for a simple system that allows for an increase in sensitivity.
A further method for this category is found in U.S. Pat. No. 4,716,106 where the primary probe sequence is first cloned in the filamentous phage M13 DNA. The single stranded form of the M13 DNA carrying the target-complementary sequence is isolated and then used as a primary probe. The DNA strand complementary to that carrying the probe is also separately isolated, labelled at multiple sites along its length and then used as the detector probe. Even though this assay involves the use of multiple labelling, this strategy necessarily involves cloning of the probe sequences in the M13 phage. Given the present molecular biology methodology, cloning of M13 is a difficult and cumbersome process, even for someone skilled in the art. The cloning must be performed every time anew in order to prepare the probe reagents directed towards a given target nucleic acid sequence. This increases the time, effort, and cost involved. Therefore, there remains a need for a simpe and sensitive assay system for detection of specific nucleic acid sequences.
Due to the complexity and involvement of each of the strategies described above, these techniques are used to a limited extent by laboratories. Therefore, a need continues to exist for a simplified, rapid, and adaptable hybridization assay wherein a primary probe hybridizes to a target and to an amplifier polynucleotide strand that allows for attachment of multiple copies of a labelling molecule.
The present invention serves to overcome the limitations of the assays currently available in the art. Those ordinarily skilled in the art will appreciate that a preferred embodiment of the present invention provides a method for amplifying a signal during the detection of target nucleic acid sequences comprising:
a) hybridizing a polynucleotide probe (the primary probe) to the target nucleic acid sequence wherein the primary probe has a means for binding to a amplification nucleic acid sequence (the amplification probe), the amplifying molecule being capable of hybridizing to at least one labelling nucleic acid sequence, conjugated to a chemical label (the labelling probe); PA1 b) immobilizing the probe-target complex; PA1 c) exposing the immobilized probe-target complex to an amplification probe (a polynucleotide sequence containing a region of multiple repeating sequence units and a region of sequences complementary to sequences in a region of the primary probe) under conditions that allow the amplification probe to hybridize to the probe-target complex; PA1 d) exposing the hybridized amplification probe to a labelling probe (a nucleic acid sequence conjugated to a chemical label and containing a region of sequences complementary to a repeating sequence unit on the amplification probe) under conditions that allow many labelling probes to hybridize to the amplification probe; PA1 e) detecting the labelling probes in the resulting complex.
There are many advantages over the prior art attained by this invention. First and foremost, the present invention greatly facilitates label detection in nucleic acid hybridization assays by providing a plurality of repeating units in the amplification probe whereby it may receive up to sixteen molecules (or more) of a selected labelling probe for every probe-target complex. This results in significantly increased sensitivity and enhanced utility of hybridization assays.
A further advantage resides in the design of the probe used in the cascade of reactions. These probes could be easily and economically synthesized with the currently available methods and instruments. The rate of hybridization reaction is much higher in this invention due to the use of many shorter probes than when longer probes are used. Moreover, use of single stranded probes also avoids the problem of self-annealing of the probes during the hybridization reaction. Since the label is not directly linked to the primary probe, the hybridization properties of primary probe are not altered, which contributes to the ability of probe reagents to be applicable in a large number of target detection assays. Therefore, the preparation of such reagents is achieved by simple methods, reducing the overall cost of the assay.
Yet a further advantage is seen in the fact that since the primary probe could always be synthesized with a poly(A) sequence, the other components used in the assay such as the amplification probe and the labelling probe could be used universally in any nucleic acid hybridization assay. This translates into a large economy of efforts, costs, and uniformity of reaction conditions. Further, this allows the possibility for quickly developing assays for any given target; the only new requirement for each project would be the construction of the specific portion of the primary probe.
A further advantage of the present invention is to provide hybridization assays having less background noise, greater sensitivity, higher signal/noise ratios, as well as greater speed than has been achievable with previously known methods. Traditionally in nucleic acid hybridization assays, the primary probe is labelled with biotin or other markers along its entire length. When required to expedite the hybridization reaction using polynucleotides, such extensive modification of the probe might lead to a reduced efficiency of its hybridization with the target molecule and therefore the probe-target complexes might not be formed rapidly or stably. Since the primary probes of these traditional assays are end labelled, each of the probe-target complexes would carry only one market and therefore the sensitivity of the assay would be limited to the detection of one marker per probe-target complex. In contrast, one embodiment of the present invention would amplify the signal be adding sixteen or more marker molecules per each probe-target complex. The sensitivity of the assay would therefore be amplified to the extent proportional to the number of marker molecules attached to the amplification probe.
In a preferred embodiment of the present invention, a method is provided for amplifying a signal during the detection of target nucleic acid sequences in a test sample containing cellular material comprising the steps of hybridizing a polynucleotide probe (the primary probe) to the target nucleic acid sequence wherein the primary probe has a means for binding to a amplification nucleic acid sequence (the amplification probe), the amplifying molecule being capable of hybridizing to at least one labelling nucleic acid sequence, conjugated to a chemical label (the labelling probe); immobilizing the probe-target complex; exposing the immobilized probe-target complex to an amplification probe (a polynucleotide sequence containing a region of multiple repeating sequence units and a region of sequences complementary to sequences in a region of the primary probe) under conditions that allow the amplification probe to hybridize to the probe-target complex; exposing the hybridized amplification probe to a labelling probe (a nucleic acid sequence conjugated to a chemical label and containing a region of sequences complementary to a repeating sequence unit on the amplification probe) under conditions that allow many labelling probes to hybridize to the amplification probe; detecting the labelling probes in the resulting complex.
In another embodiment, cells to be assayed are lysed with a lysis buffer solution and the nucleic acid sequences are denatured. The primary probe is contacted with the denatured nucleic acids in solution phase. Any probe-target complexes formed are removed from other material by immunocapture of the complexes. This is achieved by transferring the hybridization reaction mixture to microtiter plate wells that have been previously coated with monoclonal anti-probe-target complex. After washing, the immobilized probe-target complex is detected by contacting it with the amplification probe, then the labelling probe. The presence of an enzyme attached to the immobilized probe-target complex is determined by contacting the enzyme with its substrate and measuring the resulting reaction. Such an embodiment can involve the detection of Listeria monocytogenes in cheese, the detection of Escherichia coli in meat, or the detection of Salmonella typhi in human blood.